The present invention generally relates to a chimeric fibroblast growth factor protein, and in particular, to a chimeric fibroblast growth factor protein which does not have an absolute requirement for heparan sulfate for biological activity. The present invention also relates to nucleic acid molecules encoding such a protein, and to therapeutic methods of using such a protein.
Fibroblast growth factors (FGFs) comprise a growing family of proteins found throughout various organs and tissues of both developing and adult mammals. FGFs have been shown to mediate or influence numerous biological processes including mitogenesis, angiogenesis, wound healing, and neurogenesis, as well as limb patterning and outgrowth. Two particularly well known members of the FGF family include FGF-1 and FGF-2, also referred to as acidic FGF and basic FGF, respectively.
FGF-2, also referred to as basic fibroblast growth factor (bFGF), was one of the first FGFs to be identified and has been extensively studied. FGF-2 has been shown to be able to elicit various biological responses by binding to and activating specific cell-surface receptors called FGF receptor tyrosine kinases. In addition to the FGF receptor tyrosine kinase, it is generally agreed that heparan sulfate proteoglycans (or its soluble analog heparin) are necessary for both the FGF/FGF receptor interaction and the resulting biological activity. A relatively small number of studies have implicated the FGF ligand to have a role in mediating the biological activity of these factors, yet the mechanism by which this occurs remains poorly understood.
The commonly accepted paradigm for growth factor mediated activation of receptor tyrosine kinases depicts ligand-facilitated multimerization and trans-phosphorylation of the cognate receptor resulting in the recruitment of intracellular adapter and signal-transducing molecules. A complex cascade of intracellular signaling events terminating in the nucleus is thought to dictate the resulting biological response(s) (Fantl, et al., (1993) Ann. Rev. Biochem., 62:453-81; Klint, et al., (1999) Frontiers in Bioscience4: D165-77). Concomitantly, the ligand is internalized and subjected to degradation or other alternative fates (Cuatrecasas, (1982) Epidermal growth factor: uptake and fate. Ciba Foundation Symposium, 96-108; Lewis, et al., (1996)Exp. Eye Res., 62:309-24; Massagu, etal., (1986) J. Cell. Phys., 128:216-22; Naka, et al., (1993) Febs Letters, 329:147-52; Sorkin, et al., (1988) Exp. Cell Res., 175:192-205). However, mounting evidence for a number of growth factors and cytokines (FGF, nerve growth factor, PDGF, Schwannoma-derived growth factor, insulin, angiotensin 11 and growth hormone) suggest that they may act intracellularly and in many cases support a site of action for these factors in the nucleus (Jans, et al., (1998) Bioessays, 20:400-11; Prochiantz, et al., (1995) Bioessays, 17:39-44; Imamura, et al., (1990) Science, 249:1567-1570; Kimura, H. (1993) Proc. Natl Acad. Sci. USA, 90:2165-9). This has been extensively documented for the FGF family (Imamura, et al., (1990) Science, 249:1567-1570; Baldin, et al., (1990) EMBO J., 9:1511-1517; Imamura, et al., (1994) Exp. Cell Res., 215:363-372). However, the only specific activity described for FGF in the nucleus is enhancement of ribosomal RNA synthesis (Bouche, et al., (1987) Proc. Natl. Acad. Sci. USA, 84:6770-6774). This activity was also correlated with the ability of FGF-2 to bind to and regulate the activity of protein kinase CK2 which has been shown to act directly on nucleolin, a nucleolar protein involved in the control of rDNA transcription (Bonnet, et al., (1996) J. Biol. Chem., 271:24781-7). Additionally, a number of studies have shown that translocation of FGF-2 or FGF-1 to the nucleus either in the absence or presence of their cognate receptors is involved in DNA synthesis, but specific FGF targets have not been identified (Hawker, et al., (1994) Am. J. Phys., 266:H107-20; Hawker, et al., (1994) In Vitro Cellular And Developmental Biology. Animal30A:653-63; Wiedlocha, et al. (1996) Mol. Cell. Biol, 16:270-280; Wiedlocha, et al., (1994) Cell, 76:1039-1051). FGF-1 and FGF-2 ligands have been detected in intracellular compartments. Both ligands have been proposed to have specific intracellular sites of action that include stimulation of DNA synthesis for FGF-1 and stimulation of ribosomal gene transcription for FGF-2. A receptor-independent role for FGF-1 has been proposed using an FGF-1-Diphtheria toxin conjugate, which allowed receptor-independent, cytoplasmic entry of FGF-1.
The evidence for the activity of FGF proteins in a variety of beneficial biological processes, combined with the evidence indicating an intracellular site of action and a potential direct role for FGF proteins in signal transduction affecting cell proliferation and differentiation, make FGF proteins a desirable candidate molecule for the development of modified proteins as regulators of cell growth and differentiation, for the use in applications such as promoting wound healing, treating myocardial infarction (Svet-Moldavsky, G. J., et al, Lancet (Apr. 23, 1977) 913; U.S. Pat. Nos. 4,296,100 and 4,378,347), treating degenerative neurological disorders, such as Alzheimer""s disease and Parkinson""s disease (Walicke, P., et al, Proc Natl Acad Sci (USA) (1986) 83:3012-3016), promoting angiogenesis, promoting bone healing, and promoting muscle healing. Therefore, there is a need in the art for modified FGF proteins having FGF biological activity and novel attributes which improve their suitability for use in therapeutic protocols.
The present invention generally relates to a chimeric fibroblast growth factor (FGF) protein characterized by: (a) fibroblast growth factor biological activity in the absence of heparan sulfate; and, (b) an ability to enter a living cell in the absence of a receptor that binds to FGF. The present invention also relates to recombinant nucleic acid molecules encoding such a chimeric FGF protein, to therapeutic compositions including such a chimeric FGF protein, and to methods of making and using such a chimeric FGF protein.
One embodiment of the present invention is a chimeric fibroblast growth factor (FGF) which includes: (a) a biologically active fibroblast growth factor (FGF) protein having a first amino acid sequence; and, (b) a penetratin peptide having a second amino acid sequence. The penetratin peptide transports the chimeric fibroblast growth factor (FGF) across a lipid bilayer of a cell independently of the presence of an FGF receptor, and the second amino acid sequence is linked to the first amino acid sequence. The chimeric fibroblast growth factor (FGF) is characterized by: (i) fibroblast growth factor (FGF) biological activity in the absence of heparan sulfate; and, (ii) entry into a living cell in the absence of a receptor that binds to FGF. In one embodiment, the FGF biological activity of(i) is characterized by: (a) repression of terminal differentiation in the absence of heparan sulfate; and/or, (b) promotion of cell proliferation in the absence of heparan sulfate. In a preferred embodiment, the second amino acid sequence is linked to the N-terminus of the first amino acid sequence.
In the chimeric FGF of the present invention, the FGF protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule encoding any naturally occurring FGF protein, with an FGF protein selected from the group consisting of fibroblast growth factor-1 (FGF-1) protein and fibroblast growth factor-2 (FGF-2) protein being preferred. The FGF protein encoded by the nucleic acid molecule has FGF biological activity. In a preferred embodiment, the FGF protein is selected from the group consisting of a fibroblast growth factor-1 (FGF-1) protein and a fibroblast growth factor-2 (FGF-2) protein. Other preferred FGF proteins include, but are not limited to: FGF proteins having an amino acid sequence selected from the group of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. A preferred FGF protein for use in the chimera of the present invention is a fibroblast growth factor-2 protein. In one embodiment, the FGF protein has an amino acid sequence comprising from position 18 through position 172 of SEQ ID NO:2 (HLX-FGF-2) or from position 17 through 171 of SEQ ID NO:4 (TAT-FGF-2). Preferably, a biologically active FGF protein useful in a chimera of the present invention is encoded by a nucleic acid sequence comprising from nucleotide 59 to 523 of SEQ ID NO:1 (HLX-FGF-2) or from nucleotide 59 to 523 of SEQ ID NO:3.
In one embodiment, the penetratin peptide portion of a chimeric FGF of the present invention can include: (a) a first peptide having an amino acid sequence selected from the group consisting of:
(i) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16; and,
(ii) X16-X15-X14-X13-X12-X11-X10-X9-X8-X7-X6-X5-X4-X3-X2-X1;
wherein X1, X2, X3, X4, X5, X7, X8, X9, X10, X11, X12, X13, X14, X15, and X16 each represent an xcex1-amino acid, between 6 and 10 of which are hydrophobic amino acids; and wherein X6 represents Trp; and,
(b) a second peptide comprising amino acid residues 49-57 of HIV Tat protein (SEQ ID NO:17). In a preferred embodiment, the second peptide of (b) does not comprise amino acid residues 22-36 or 73-86 of HIV Tat protein (SEQ ID NO:17).
The first penetratin peptide can include a peptide comprising helix 3 of a homeobox domain and a homeobox domain, and fragments and homologues thereof Such peptides comprise an amino acid sequence including, but are not limited to: SEQ ID NO:9, amino acid residues 42 through 58 of SEQ ID NO:9, amino acid residues 43 through 59 of SEQ ID NO:9, amino acid residues 43 through 58 of SEQ ID NO:9, amino acid residues 58 through 43 of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and/or SEQ ID NO:16. In one embodiment, such a peptide comprises amino acid residues 2-17 of SEQ ID NO:2. Preferably, such a peptide is encoded by a nucleic acid sequence comprising nucleotides 11 to 58 of SEQ ID NO:1.
The second penetratin peptide can include an HIV Tat protein or fragments or homologues thereof. Preferred peptides comprise an amino acid sequence that includes, but is not limited to: amino acid residues 37-72 of SEQ ID NO:17, amino acid residues 38-72 of SEQ ID NO:17, amino acid residues 47-72 of SEQ ID NO:17, amino acid residues 37-58 of SEQ ID NO:17, amino acid residues 38-58 of SEQ ID NO:17, amino acid residues 47-58 of SEQ ID NO:17, amino acid residues 1-21 and 38-72 of SEQ ID NO:17, amino acid residues 47-62 of SEQ ID NO:17, amino acid residues 38-62 of SEQ ID NO:17, amino acid residues 1-72 of SEQ ID NO:17, amino acid residues 1-58 of SEQ ID NO:17, and/or amino acid residues 48-60 of SEQ ID NO:17. In one embodiment, such a peptide comprises amino acid residues 48-60 of SEQ ID NO:17 or amino acid residues 2-14 of SEQ ID NO:4. Preferably, such a peptide is encoded by a nucleic acid sequence comprising residues 14 to 52 of SEQ ID NO:3.
A chimeric fibroblast growth factor (FGF) of the present invention includes a chimera comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2 (HLX-FGF-2) and SEQ ID NO:4 (TAT-FGF-2). Preferably, such a chimeric FGF is encoded by a recombinant nucleic acid molecule having a nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:3, respectively.
Another embodiment of the present invention relates to a therapeutic composition comprising the chimeric fibroblast growth factor (FGF) of the present invention and a pharmaceutically acceptable excipient.
Yet another embodiment of the present invention relates to a recombinant nucleic acid molecule encoding a chimeric fibroblast growth factor (FGF) of the present invention as described above. Such a recombinant nucleic acid molecule comprises: (a) a first isolated nucleic acid sequence encoding a biologically active fibroblast growth factor (FGF) protein; and, (b) a second isolated nucleic acid sequence encoding a penetratin peptide that transports the chimeric fibroblast growth factor (FGF) across a lipid bilayer of a cell independently of the presence of an FGF receptor, wherein the second nucleic acid sequence is linked to the first nucleic acid sequence. The first and second nucleic acid sequences are operatively linked to a transcription control sequence. Such a chimeric fibroblast growth factor (FGF) is characterized by: (i) fibroblast growth factor biological activity in the absence of heparan sulfate; and, (ii) entry into a living cell in the absence of a receptor that binds to FGF. Preferred chimeric FGF proteins encoded by a recombinant nucleic acid molecule of the present invention are described above.
Another embodiment of the present invention relates to a recombinant cell that expresses the recombinant nucleic acid molecule of the present invention described above. Another embodiment of the present invention is a recombinant virus that comprises the recombinant nucleic acid molecule of the present invention.
Yet another embodiment of the present invention relates to a method to produce a chimeric fibroblast growth factor (FGF), comprising culturing in an effective medium a recombinant cell comprising a recombinant nucleic acid molecule encoding a chimeric fibroblast growth factor protein as described above.
Another embodiment of the present invention relates to a method to promote fibroblast growth factor biological activity in a cell and particularly, to repress terminal differentiation and promote proliferation in a cell. Such a method includes the steps of administering to a cell a chimeric fibroblast growth factor (FGF) protein of the present invention as described above. In one embodiment, the cell has reduced heparan sulfate proteoglycan production characterized by a reduction in both repression of terminal differentiation and promotion of proliferation in the presence of naturally occurring fibroblast growth factor. In another embodiment, the cell is a cell of patient that has a condition selected from the group consisting of stroke, nerve damage, bone damage, muscle damage, and a wound. Such a chimeric FGF can be administered by any route, including in vitro, in vivo, and ex vivo.
Another embodiment of the present invention relates to a method to enhance a biological process selected from the group consisting of mitogenesis, angiogenesis, wound healing, neurogenesis, limb patterning, limb outgrowth, comprising administering to cells associated with the biological process a chimeric fibroblast growth factor (FGF) of the present invention as described above.